The present invention relates generally to imaging systems and methods, and more particularly to high resolution microscopy systems and methods using optical amplification.
For over a century the resolution of far-field optical microscopes has been limited by the Abbe diffraction limit. This limit applies to both wide-field and laser-scanning microscopes. For the best of current-generation far-field optical microscopes, the resolution limit is around 200 nm in the lateral x and y directions (perpendicular to the beam path) and about 500 nm in the axial z direction (along the beam path). The axial resolution in the z direction is worse than the transverse resolution limit because of the diffraction of the light beam crossing the objective lens. Near-field optical microscopy can reach a lateral resolution better than 100 nm, but it is confined to imaging a surface within the vicinity of the evanescent optical near field. Thus, far-field optical microscopes remain a good option for imaging many 3D structures.
A laser-scanning confocal microscope has the ability to generate 3D images through high-resolution axial sectioning. Such a microscope has a much better depth range than a wide-field microscope of the same resolution, but conventional confocal microscopes are also limited by the Abbe diffraction limit.
A 4 Pi confocal microscope provides an improved resolution in the z direction over that of a conventional confocal microscope by focusing the light with two opposing high numerical aperture (NA) objective lenses to create two interfering spherical waves, which result in a spherical spot (see, e.g., S. Hell and E. H. K. Stelzer, “Properties of a 4 Pi confocal fluorescence microscope,” J. Opt. Soc. Am. A 9, 2159-2166 (1992)). Even with this improvement, the resolution of a 4 Pi microscope is also diffraction-limited.
Nonlinear techniques can break the diffraction limit. Recently, it was demonstrated that it is possible to narrow the focal spot of a fluorescence microscope below the diffraction limit by applying the highly nonlinear process of stimulated emission, a technique known as stimulated emission depletion (STED) (see, e.g., T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Arad Acad. Sci. 97, 8206-8210 (2000)). In STED techniques, a long-wavelength de-excitation pulse follows immediately after a short-wavelength excitation pulse. The de-excitation pulse contains a zero in its spatial intensity profile at the beam center and is aligned around the excitation focal point in a ring structure so that the molecules in the center of the ring are excluded from de-excitation. Saturation depletion by the de-excitation pulse dramatically reduces the fluorescent spot in the center to a transverse subdiffraction size that is not limited by the wavelength, but only by the applicable pulse intensity. A transverse resolution down to 16 nm in the focal plane, corresponding to about 1/50 of the STED wavelength of 775 nm was recently accomplished (see V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94, 143903 (2005)). If conventional confocal imaging is used for a STED microscope, however, the axial resolution in the z direction is still limited to about ½ of the wavelength.
A combination of STED with 4 Pi microscopy has lead to a resolution of 30-50 nm in the z direction (see, e.g., M. Dyba and S. W. Hell, “Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88, 163901 (2002)). However, existing 4 Pi schemes, which use two opposing high NA objective lenses that focus at the same spot, are expensive and very difficult to align. Also, STED requires the use of two ultrashort (e.g., picosecond or femtosecond) laser pulses, one at the excitation wavelength and another at the STED wavelength. The pulses have to be synchronized for the STED pulse to follow the excitation pulse at an optimum delay, and the spatial phase of the STED pulse has to be manipulated through special optics so that it is focused into a spatial profile that has a zero at the center. Furthermore, the focused STED pulse has to be carefully aligned with the excitation pulse so that the zero at its center overlaps with the peak of the excitation spot exactly at nanometer resolution. As a consequence, STED is also very expensive and difficult to implement.
Therefore it is desirable to provide systems and methods that overcome the above and other problems.